Method and system for optical force measurement

ABSTRACT

One aspect of this disclosure relates to a computer-implemented method for determining a force acting on at least part of a structure, for example a biological structure, such as a DNA molecule. The method comprises controlling a light-sensitive system, e.g. of a microscope, to determine light information based on light from the structure. The light is incident on at least a part of the light sensitive system. The light-sensitive system may be said to capture the light from the structure. The at least part of the structure comprises one or more optically active entities, such as DNA intercalator molecules and donor/acceptor fluorophores. At least one of (i) an optical activity of the entities and (ii) a quantity of the entities depends on the force acting on the at least part of the structure. Furthermore, the light information defines a light property value associated with said at least part of the structure. The method further comprises determining the force acting on the at least part of the structure on the basis of said light property value and a reference light property value.

FIELD OF THE INVENTION

This disclosure relates to a method, a controller and a computer programfor determining a force acting on at least part of a structure based onlight from the structure.

BACKGROUND

The ability to sensitively detect forces acting on and/or withinbiological structures, such as DNA, proteins and cells, is of greatimportance as a means to understand the mechanics and physicalinteractions that govern biological function. For example, in proteinunfolding experiments the forces involved are monitored in order todetermine the mechanical force at which a protein domain unfolds. Asanother example, in the field of molecular motors, it is examined howenzymatic reactions can generate force to drive directed motion along asubstrate of DNA or to transport cargo along the cytoskeleton. In thefield of DNA origami, molecular structures are synthesized that haveinternal tension or of which the mechanical properties are important fortheir designed function, such as to study the interacting forces betweenDNA and proteins. In force measurement experiments, typically forcespectroscopy techniques are utilized, for example involving opticaltweezers holding beads that are bound to a structure of interest. Thetraps holding the beads are usually calibrated which enables todetermine a force that causes a movement of a bead within the trap.Disadvantageously, such experiments require the use of advancedtechnology.

Experiments involving force spectroscopy techniques are described in“Biebricher, A. S. et al. The impact of DNA intercalators on DNA andDNA-processing enzymes elucidated through force-dependent bindingkinetics. Nat. Commun. 6:7304 doi: 10.1038/ncomms8304 (2015)”.Biebricher discloses experiments that aim to elucidate the impact of DNAintercalator molecules on DNA and DNA-processing enzymes. DNAintercalator molecules are namely widely used as fluorescent probes tovisualize and study DNA processes. To this end, a tension-dependentbinding constant is determined based on measured force-elongationcurves. For obtaining these curves, the applied forces are measuredusing conventional back-focal plane interferometry and the associatedelongations are measured using either conventional camera tracking orare calculated based on fluorescence intensity measurements.

Wang and Ha, “Defining Single Molecular Forces Required to ActivateIntegrin and Notch Signaling”, Science 340, 991-994 (2013) describe amethod for estimating the molecular forces that are required foractivating intracellular signalling using tension-gauge tethers. Herein,a ligand for a membrane receptor is immobilized on a surface through atether, which ruptures if a force higher than a critical force isapplied to it. When a receptor of a molecule engages with and appliestension to the ligand, the tether thus may or may not rupture. If signalactivation through the receptor requires a molecular force larger thanthe critical force of the tether, it will rupture, abolishing signalactivation. In contrast, if the required force is smaller than thecritical force, the tether will endure, activating the receptor-mediatedsignalling. By engineering a series of tethers that rupture at differentforces, the force required for signal activation can be determined.

Disadvantageously, this method requires multiple tethers respectivelyassociated with different critical forces to be engineered. Furthermore,the method only allows to determine that an applied force is within arange between two consecutive critical forces associated with tworespective tethers. However, it is difficult to construct a series oftethers such that they have densely and precisely defined criticalforces. Hence, the accuracy of this method is limited. Further, thismethod is not suitable for continuously measuring a force that varieswith time. In an example, a force initially increases to a maximum forceand then decreases. In such case, once the force has reached itsmaximum, the tethers having a critical force lower than the maximumforce will have been ruptured. As a result, no means are left to measurethe decrease of the force.

Hence, there is a need in the art for an improved method for measuringforces applied to structures that alleviates at least some of theproblems identified above.

SUMMARY

Therefore, one aspect of this disclosure relates to acomputer-implemented method for determining a force acting on at leastpart of a structure, for example a biological structure, such as a DNAmolecule. The method comprises controlling a light-sensitive system,e.g. a light-sensitive system of a microscope, to determine lightinformation based on light from the structure. The light is incident onat least a part of the light sensitive system. The light-sensitivesystem may be said to capture the light from the structure.

The at least part of the structure comprises one or more opticallyactive entities, such as DNA intercalator molecules and donor/acceptorfluorophores. At least one of (i) an optical activity of the entitiesand (ii) a quantity of the entities depends on the force acting on theat least part of the structure. Furthermore, the light informationdefines a light property value associated with said at least part of thestructure. The method further comprises determining the force acting onthe at least part of the structure on the basis of said light propertyvalue. Optionally, the method comprises determining the force based on areference light property value as well. The reference light propertyvalue may be pre-stored in a data storage and may be associated with areference force.

One distinct aspect of this disclosure relates to a method for causingat least part of a structure to exhibit a force dependent opticalactivity for enabling determination of a force acting on the at leastpart of the structure in accordance with one or more of the methods fordetermining a force as described herein. The method comprises combiningthe at least part of the structure, a fluid and optically activeentities. The optically active entities in the fluid can bind to the atleast part of the structure. A binding property in respect of the atleast part of the structure depends on the force acting on the at leastpart of the structure thus causing that the quantity of the entitiescomprised by the structure depends on the force acting on the structure.Additionally or alternatively to a binding property, an optical propertyof optically active entities bound to the at least part of the structuredepends on the force acting on the at least part of the structure.

Optionally an optical property, such as a quantum yield, of an opticallyactive entity depends on whether the entity is bound or unbound to theat least part of the structure.

It should be appreciated that the above-mentioned method for determiningthe force acting on the at least part of the structure optionallycomprises one or more of the method steps of methods described hereinfor causing at least part of the structure to exhibit a force dependentoptical activity.

The methods disclosed herein enable to determine a force acting on astructure without the need to engineer multiple tethers that rupture atdifferent critical forces. Advantageously, the disclosed method furtherdoes not require complex force detection systems, such as systemscomprising calibrated optical or acoustical traps, that may also requireattaching microscopic beads to the structure, as is typically done inoptical tweezer experiments. The disclosed method enables to measure themagnitude of forces using relatively simple experimental set-ups andgeneral purpose equipment. Hence, the technologies disclosed hereinobviate the need to build complex force detection systems and/or attachmicroscopic beads as is typically done in e.g. optical tweezerexperiment. Since at least one of a quantity and an optical property ofthe optically active entities depends on the force acting on thestructure, the at least part of the structure exhibits a force-dependentoptical activity and hence, a property of the captured light isrelatable to the magnitude of the acting force. Such property, and thusthe optical activity and/or the light property value may relate to atleast one of a light intensity, e.g. fluorescence intensity, light colorand light polarization. In principle, an arbitrarily small change inforce, will result in a change in the light property value as defined bythe image data. In contrast, in force measurements using tension-gaugetethers, a force change will not necessarily result in a rupture of atether. In such case, the force change will remain unnoticed. Thedisclosed methods herein thus provide an enhanced accuracy. The methodfurther provides the advantage that the force may be continuouslymeasured, since the method does not depend on the irreversible ruptureof tethers, yet, for example, on the reversible increase and decrease offluorescence. For example, the method enables to measure the varyingmagnitude of forces acting on a DNA molecule in real-time. Furthermore,the method enables to simultaneously measure forces that are acting ondifferent structures or different parts of a structure that are presentwithin the field of view of a fluorescence microscope.

In one embodiment, the structure comprises, e.g. consists of and/or is,a DNA molecule. This embodiment advantageously allows to study theforces acting on and/or within DNA molecules.

In one embodiment, the method comprises controlling a force applicationsystem to apply a force to the at least part of the structure. Inparticular, in one embodiment, the method comprises controlling a forceapplication system to apply a further force to the at least part of thestructure on the basis of the determined force. It should be appreciatedthat the force application system is not necessarily calibrated. Anexample of such a system would be a structure manipulation system fordeforming, e.g. extending, the structure. In such a system, adeformation may be controlled, as a result of which a force is appliedto the structure. This embodiment enables to both apply a force to thestructure and subsequently measure the applied force. This measurementmay then be used to adjust the applied force. Such a feedback loopenables to accurately apply forces to the structure without using any ofa calibrated optical trap, an atomic force microscope or similarcalibrated equipment.

In one embodiment, the method comprises controlling a force applicationsystem to change the force acting on the at least part of the structure.The embodiment comprises controlling the light-sensitive system tocapture light from the structure while said force is changing. The lightinformation defines, for a plurality of time instances, a light propertyvalue associated with said at least part of the structure. Theembodiment further comprises, for each time instance, based on the lightproperty value, determining the force acting on the at least part of thestructure. This embodiment enables to continuously monitor the forceacting on the at least part of the structure.

In one embodiment, the light-sensitive system comprises an imagingsystem and the light information comprises image data, that may bespatially resolved. The image data define a set of light property valuesassociated with respective parts of the structure. In this embodimentthe method further comprises, for each part, based on its associatedlight property value, determining a force acting on the part of thestructure. This embodiment advantageously enables to measure the forcesacting on different parts of the structure. This embodiment for exampleenables to measure the internal forces along extended moleculararchitectures, e.g. along the length of a DNA molecule. Hence, themagnitudes of local forces, in particular intra-DNA forces, may bemeasured. Such detailed force profiles enable to accurately study themechanical and physical properties of dsDNA.

In one embodiment, the light-sensitive system comprises an imagingsystem and the light information comprises image data. The image datarepresent an image of the at least part of the structure and comprise aset of image pixel values associated with respective parts of thestructure. In this embodiment, determining the force comprisesdetermining one or more subsets of one or more image pixel values. Eachsubset defines a region of interest (ROI) in the image. Determining theforce further comprises, for each ROI, determining the force acting on apart of the structure represented by the ROI based on the image pixelvalues defining the ROI. The image pixel values may represent the lightproperty value as described herein. This embodiment also enables tomeasure the forces acting on different parts of the structure. Thisembodiment further allows for efficient processing of obtained imagedata and enables flexibility in determining an area of the structure onwhich an average force may be determined. In an example, the one or moresubsets may be determined in response to a user input. A user may, whenthe image is rendered on a display, select the regions of interest inthe image for which a force is to be determined. In this example, auser, by selecting a region of interest in the image, which region ofinterest represents a part of the structure, he selects a subset of oneor more image pixel value based on which a force acting on the partrepresented by the region of interest is to be determined.

In one embodiment, the method comprises receiving a user input selectingan initial subset of image pixel values and the method further comprisesdetermining the subset of image pixel values by trimming theuser-selected subset of image pixel values, for example based on acriterium that the subset of image pixel value may comprise only imagepixel values above or below a threshold value. This would allow a userto roughly select a subset of image pixel values, for example by“drawing” an area in an image presented to him, wherein the areacomprises a representation of a part of the structure that is ofinterest to the user. Subsequently, the method may then compriseautomatically selecting the image pixel values that actually correspondto the structure, and disregard the image pixel values within said areathat do not correspond to the structure, which values may distort theforce determination. In an example such disregarded image pixel valuescorrespond to dark background image pixels.

In one embodiment, the light-sensitive system comprises an imagingsystem and the light information comprises image data. The image datadefine a set of light property values associated with respective pluralstructures, one of these structures being the above-mentioned structure.In this embodiment, the method further comprises, for each structure,based on its associated light property value, determining a force actingon the structure.

In this embodiment, in particular, the image data may define a set oflight property values associated with respective parts of pluralstructures, one of these structures being the above-mentioned structure.Then, the method further comprises, for each part, based on itsassociated light property value, determining a force acting on the part.

In this embodiment, the plural structures may be similar structures. Inan example these structures comprises or are DNA molecules. Theseembodiments enable to simultaneously measure forces acting on differentstructures and/or on different parts of different structures.

In one embodiment, the method comprises the steps of obtaining aparameter relating a difference of force magnitude to a difference inoptical property value. In this embodiment the method also comprisesdetermining the force acting on the at least part of the structure basedon said parameter. The parameter may be pre-stored in a data storageconnected to a controller. This embodiment thus advantageously enablesto determine a difference between forces without requiring theapplication of known reference forces. The embodiment enables todetermine a difference between two forces, even if these two forces areunknown.

In one embodiment, the method comprises controlling the light-sensitivesystem to determine reference light information based on reference lightfrom a reference structure while a reference force is acting on at leasta part of the reference structure. The reference light is incident on atleast a part of the light-sensitive system. The at least part of thereference structure comprises one or more optically active referenceentities. At least one of an optical activity of the reference entitiesand a quantity of the reference entities depends on the reference forceacting on the at least part of the reference structure. The referencelight information defines the reference light property value that isassociated with said at least part of the reference structure.

The reference structure may be the same structure as the structure onwhich the to be determined force is acting, “the structure of interest”.This allows to both calibrate the force measurement and measure theforce using a single structure, wherein the calibration and themeasurement may be performed in one experiment or different experiments.In case the reference structure is the same structure, the at least partof the reference structure may be the same as or different from the atleast part of the structure of interest. In an example, the same part ofthe structure is analyzed twice subsequently. One time for calibratingthe force measurement method and another time for determining a forceacting on that part of the structure.

The reference structure may be a different structure from the structureof interest. This allows to perform a separate experiment on a separatestructure for calibration. In yet another example, the referencestructure may be imaged together with the structure of interest during asingle experiment. This embodiment enables an absolute determination ofthe force applied to the at least part of the structure.

In one embodiment, the method comprises controlling the light sensitivesystem to determine second reference light information based on secondreference light from a second reference structure while a secondreference force is acting on at least a part of the second referencestructure. The second reference light is incident on at least a part ofthe light-sensitive system. The at least part of the second referencestructure comprises one or more optically active second referenceentities. At least one of an optical activity of the second referenceentities and a quantity of the second reference entities depends on thesecond reference force acting on the at least part of the secondreference structure. The second reference light information defines asecond reference light property value associated with said at least partof the second reference structure. In this embodiment, the methodcomprises determining the force acting on the at least part of thestructure based on the second reference light property value.

In this embodiment, the step of determining the force may comprisedetermining, based on the reference light property value and secondreference light property value, the parameter relating a difference offorce magnitude to a difference in optical property value anddetermining the force acting on the at least part of the structure basedon this parameter.

The second reference structure may or may not be the same as thestructure of interest and may or may not be the same as the referencestructure mentioned above. The at least part of the second referencestructure may or may not be the same as the at least part of thestructure and may or may not be the same as the at least part of thestructure of interest.

This embodiment advantageously enables to experimentally determine theabove-mentioned parameter relating a difference of force magnitude to adifference in light property value. Furthermore, this embodiment enablesto obtain reference data associating a plurality of forces with aplurality of light property values, respectively. Then, the force may bedetermined based on these reference data.

In one embodiment, the structure is at least partially positioned in afluid comprising optically active entities, such as DNA intercalators,e.g. cyanine intercalators, wherein a binding property of the opticallyactive entities in respect of the at least part of the structure dependson the force acting on the at least part of the structure.

The fluid may further comprise additives, such as e.g. oxygen scavengersto reduce photobleaching or salts to regulate the physical properties ofthe at least part of the structure, and/or its interaction withoptically active entities.

In this embodiment, the force-dependency of the optical activity of thestructure is caused in an efficient manner. This embodiment allows tostudy local force profiles along a part of the structure without using acalibrated trap. In an example the local force profile along a structuremay be determined, wherein the structure is tethered on only one side.

In one embodiment, additionally or alternatively to a binding propertydepending on force, other parameters of the optically active entitiesdepend on the force acting on the at least part of the structure. In anexample, the optically active entities exhibit an increased or decreasedmobility, such as a thermal fluctuation, with increasing force appliedto the at least part of the structure. In another example, a blinking ofthe optically active entities is dependent on the force on thestructure.

In one embodiment, while the reference light is incident on at least apart of the light-sensitive system, a first concentration of opticallyactive entities is present in the fluid. In this embodiment, the methodfurther comprises controlling the light-sensitive system to determinesecond reference light information based on second reference light fromthe reference structure while the reference force is acting on at leasta part of the reference structure. The second reference light beingincident on at least a part of the light-sensitive system. While thesecond reference light is incident on the at least a part of thelight-sensitive system, a second concentration of optically activeentities different from the first concentration is present in the fluid.The second reference light information defines a second reference lightproperty value associated with said at least part of the referencestructure. In this embodiment, the method comprises determining theforce acting on the at least part of the structure based on the secondreference light property value. Advantageously this method enables toeasily calibrate the method. Only a concentration of entities in thefluid needs to change, while an applied reference force is keptconstant.

In one embodiment, the structure is connected to another structure andthe force acting on at least part of the structure is exerted by theother structure on the structure. This embodiment can be employed toreport forces in any biological system that can be mechanically coupledto the structure, e.g. coupled to a dsDNA linker molecule. Furthermore,this embodiment allows to investigate a mechanical response of the otherstructure.

This embodiment may further comprise controlling a force applicationsystem to apply a force to the other structure. Again, the forceapplication system may be controlled based on a previously determinedforce. This embodiment thus provides a straightforward method forstudying the mechanical response of the other structure, for example tostudy force-induced conformational changes (e.g. protein unfolding).

In one embodiment, the structure is at least partially positioned in aflow cell, and wherein the force acting on at least part of thestructure comprises a drag force caused by a fluid flow.

In order to apply flow-stretch assays to study DNA-protein interactions,for example, it can be highly advantageous to quantify how tensionvaries along the length of a DNA substrate, for example becauseinhomogeneous tension can alter DNA-protein interactions or otherreactions involving DNA. Often, however, it is either undesirable orimpractical to tether a bead to the DNA. Tethering a bead to DNAconstitutes an additional experimental step and may cause restrictions.There may be a risk of tethering too many DNA molecules to one bead.Furthermore, there may be a need to have two different labels on eitherend of DNA, which may be problematic if the range of DNA material for anexperiment is limited. Typically in flow-based assays (e.g. DNAcurtains), the DNA is tethered on both ends to a surface. In this case,it is physically impossible to tether a free end of the DNA to a bead.In other cases (e.g. hydrodynamic traps) DNA is free in solution andthus attaching a bead to the DNA may perturb the DNA's dynamics in thesolution. In these cases, the force applied to the DNA can only beestimated based on its apparent extension. Even if it is possible totether a bead to the structure in a flow-based assay, it is not trivialto measure a force with it due to angles and proximity surface and timeresolution. This embodiment thus enables a straightforward and sensitivemeans to determine the drag force and/or a drag force profile on DNA inany flow-based assay.

In one embodiment, the structure is at least partially positioned in aholographic optical trap. This embodiment advantageously enables asimple method for determining a force on the structure when it is aholographic optical trap, for which known force measurement methods areless suitable.

In one embodiment, the optically active entities comprise at least onepair, preferably a plurality of pairs, of a donor fluorophore and anacceptor fluorophore. The at least one pair exhibits an emissionspectrum that depends on the force acting on the structure, for examplebecause the donor and acceptor entities are connected to a mechanicalbackbone, such as a DNA molecule which acts as a spring, i.e. it ensuresthat a force that is applied to the backbone causes the average distancebetween donor and acceptor moieties to increase or decrease with theapplied force. In an example, a Förster resonance energy transfer (FRET)can occur between the donor and acceptor. This embodiment enables thatthe structure possesses intrinsic optical properties that areforce-dependent. Furthermore, this embodiment enables to determine aforce based on a color of the light coming from the structure. As knownin the art, a DNA molecule can be synthesized using fluorescent baseanalogues of the standard nucleobases (A, G, C, T and U), whichfluorescent base analogues can function as donor and acceptorfluorophores. By specifying a sequence of a DNA, the average distancebetween donor and acceptor bases can be tuned to optimize the forcesensitivity and dynamic range of the probe.

One distinct aspect of this disclosure relates to a method for causingat least part of a structure to exhibit a force-dependent opticalactivity for enabling determination of a force acting on the at leastpart of the structure in accordance with one or more of the methods asdescribed herein. This method comprises binding at least one pair of adonor fluorophore and acceptor fluorophore as described herein to thestructure.

One embodiment relates to a method for determining a force acting on atleast part of a DNA molecule. This embodiment comprises controlling animaging system of a fluorescence microscope to determine lightinformation based on fluorescent light from the structure. Thefluorescent light is incident on at least a part of the imaging system.The at least part of the DNA molecule comprises one or more DNAintercalator molecules, wherein a quantity of the molecules bound to theDNA molecule depends on the force acting on the at least part of the DNAmolecule and wherein the light information defines a light intensityvalue associated with said at least part of the DNA molecule. Thisembodiment further comprises determining the force acting on the atleast part of the DNA molecule on the basis of said light intensityvalue and a reference light intensity value.

One embodiment relates to a method for causing at least part of a DNAmolecule to exhibit a force dependent fluorescence for enablingdetermination of a force acting on the at least part of the DNA moleculein accordance with one or more of the methods for determining a force asdescribed herein. The embodiment comprises combining the at least partof the DNA molecule with a fluid and fluorescent DNA intercalatormolecules. The DNA intercalator molecules in the fluid can bind to theat least part of the DNA molecule. A binding property of the DNAintercalator molecules in respect of the at least part of the DNAmolecule depends on the force acting on the at least part of the DNAmolecule. Herein, preferably, also a fluorescence quantum yield of a DNAintercalator molecule depends on whether the DNA intercalator moleculeis bound or unbound to the at least part of the DNA molecule, whichyields a reduction of background light.

One aspect of this disclosure relates to a controller, wherein thecontroller is configured to perform one or more of the steps asdescribed herein that relate to controlling the light-sensitive systemand/or determining the force and/or controlling the force applicationsystem and/or determining one or more subsets of image pixel valuesand/or obtaining the parameter and/or any step that can becomputer-implemented.

One distinct aspect of this disclosure relates to a computer programcomprising instructions which, when the program is executed by acomputer, cause the computer to carry out one or more of the steps ofthe methods as described herein.

One distinct aspect of this disclosure relates to a computer-readablestorage medium comprising instructions which, when executed by acomputer, cause the computer to carry out one or more of the methodsteps as described herein.

As will be appreciated by one skilled in the art, aspects of the presentinvention may be embodied as a system, a method or a computer programproduct. Accordingly, aspects of the present invention may take the formof an entirely hardware embodiment, an entirely software embodiment(including firmware, resident software, micro-code, etc.) or anembodiment combining software and hardware aspects that may allgenerally be referred to herein as a “circuit,” “module” or “system.”Functions described in this disclosure may be implemented as analgorithm executed by a processor/microprocessor of a computer.Furthermore, aspects of the present invention may take the form of acomputer program product embodied in one or more computer readablemedium(s) having computer readable program code embodied, e.g., stored,thereon.

Any combination of one or more computer readable medium(s) may beutilized. The computer readable medium may be a computer readable signalmedium or a computer readable storage medium. A computer readablestorage medium may be, for example, but not limited to, an electronic,magnetic, optical, electromagnetic, infrared, or semiconductor system,apparatus, or device, or any suitable combination of the foregoing. Morespecific examples of a computer readable storage medium may include, butare not limited to, the following: an electrical connection having oneor more wires, a portable computer diskette, a hard disk, a randomaccess memory (RAM), a read-only memory (ROM), an erasable programmableread-only memory (EPROM or Flash memory), an optical fiber, a portablecompact disc read-only memory (CD-ROM), an optical storage device, amagnetic storage device, or any suitable combination of the foregoing.In the context of the present invention, a computer readable storagemedium may be any tangible medium that can contain, or store, a programfor use by or in connection with an instruction execution system,apparatus, or device.

A computer readable signal medium may include a propagated data signalwith computer readable program code embodied therein, for example, inbaseband or as part of a carrier wave. Such a propagated signal may takeany of a variety of forms, including, but not limited to,electro-magnetic, optical, or any suitable combination thereof. Acomputer readable signal medium may be any computer readable medium thatis not a computer readable storage medium and that can communicate,propagate, or transport a program for use by or in connection with aninstruction execution system, apparatus, or device.

Program code embodied on a computer readable medium may be transmittedusing any appropriate medium, including but not limited to wireless,wireline, optical fiber, cable, RF, etc., or any suitable combination ofthe foregoing. Computer program code for carrying out operations foraspects of the present invention may be written in any combination ofone or more programming languages, including an object orientedprogramming language such as Java(™), Smalltalk, C++ or the like andconventional procedural programming languages, such as the “C”programming language or similar programming languages. The program codemay execute entirely on the user's computer, partly on the user'scomputer, as a stand-alone software package, partly on the user'scomputer and partly on a remote computer, or entirely on the remotecomputer or server. In the latter scenario, the remote computer may beconnected to the user's computer through any type of network, includinga local area network (LAN) or a wide area network (WAN), or theconnection may be made to an external computer (for example, through theInternet using an Internet Service Provider).

Aspects of the present invention are described below with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems), and computer program products according to embodiments of thepresent invention. It will be understood that each block of theflowchart illustrations and/or block diagrams, and combinations ofblocks in the flowchart illustrations and/or block diagrams, can beimplemented by computer program instructions. These computer programinstructions may be provided to a processor, in particular amicroprocessor or a central processing unit (CPU), of a general purposecomputer, special purpose computer, or other programmable dataprocessing apparatus to produce a machine, such that the instructions,which execute via the processor of the computer, other programmable dataprocessing apparatus, or other devices create means for implementing thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

These computer program instructions may also be stored in a computerreadable medium that can direct a computer, other programmable dataprocessing apparatus, or other devices to function in a particularmanner, such that the instructions stored in the computer readablemedium produce an article of manufacture including instructions whichimplement the function/act specified in the flowchart and/or blockdiagram block or blocks.

The computer program instructions may also be loaded onto a computer,other programmable data processing apparatus, or other devices to causea series of operational steps to be performed on the computer, otherprogrammable apparatus or other devices to produce a computerimplemented process such that the instructions which execute on thecomputer or other programmable apparatus provide processes forimplementing the functions/acts specified in the flowchart and/or blockdiagram block or blocks.

The flowchart and block diagrams in the figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams may represent a module, segment, or portionof code, which comprises one or more executable instructions forimplementing the specified logical function(s). It should also be notedthat, in some alternative implementations, the functions noted in theblocks may occur out of the order noted in the figures. For example, twoblocks shown in succession may, in fact, be executed substantiallyconcurrently, or the blocks may sometimes be executed in the reverseorder, depending upon the functionality involved. It will also be notedthat each block of the block diagrams and/or flowchart illustrations,and combinations of blocks in the block diagrams and/or flowchartillustrations, can be implemented by special purpose hardware-basedsystems that perform the specified functions or acts, or combinations ofspecial purpose hardware and computer instructions.

Moreover, a computer program for carrying out the methods describedherein, as well as a non-transitory computer readable storage-mediumstoring the computer program are provided. A computer program may, forexample, be downloaded (updated) to the existing systems or be storedupon manufacturing of these systems.

Elements and aspects discussed for or in relation with a particularembodiment may be suitably combined with elements and aspects of otherembodiments, unless explicitly stated otherwise. Embodiments of thepresent invention will be further illustrated with reference to theattached drawings, which schematically will show embodiments accordingto the invention. It will be understood that the present invention isnot in any way restricted to these specific embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the invention will be explained in greater detail byreference to exemplary embodiments shown in the drawings, in which:

FIG. 1A depicts a system according to one embodiment;

FIG. 1B depicts method steps according to one embodiment;

FIG. 2A depicts an embodiment of the system comprising a forceapplication system;

FIG. 2B depicts a method according to an embodiment comprisingcontrolling the force application system;

FIG. 3A visualizes obtained image data according to an embodiment;

FIG. 3B illustrates calibration results according to one embodiment;

FIG. 4A visualizes image data according to one embodiment;

FIGS. 4B and 4C illustrate force measurement results according to oneembodiment;

FIG. 5 illustrates set-up and results of a force measurement accordingto an embodiment;

FIG. 6 illustrates set-up and results of a force measurement accordingto an embodiment;

FIG. 7 schematically shows a set-up for performing a force measurementaccording to one embodiment involving another structure;

FIG. 8 shows a data processing system according to one embodiment.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a system 100 for determining a force acting on at leastpart of a structure 110. The structure may consist of and/or be a DNAmolecule. The system 100 comprises a light sensitive system 102 and acontroller 104 that is configured to control the system and perform oneor more steps, for example steps S104, S106 and S106 described withreference to FIG. 1B. In this embodiment, the light sensitive system 102is positioned such that a light-sensitive part of the system 102, e.g.an imaging plane, receives light 106 from the structure 110.

The light-sensitive system 102 may be part of a microscope, for examplea fluorescence microscope. The microscope for example comprises a lightsource, such as a laser. The system may further comprise an opticalsystem for directing light from the light source to the sample 108, inparticular to the at least part of the structure 110. The light from thelight source may excite the at least part of the structure, for exampleexcite entities, e.g. fluorescent entities, bound to the at least partof the structure. As a result, the entities may emit light 106, e.g.fluorescent light. The system 100 may further comprise a second opticalsystem for directing the light emitted by the entities to thelight-sensitive system 102, which may be an imaging system 102, such asa CCD camera.

The structure 110 may thus be positioned in a sample or sample holder108. The light 106 may be emitted by at least one of the structure 110and the optically active entities bound to the structure 110 (notshown). However, the light 106 from the structure 110 may also be lightthat has passed through the structure. In the latter case, the opticalactivity of the optically active entities for example relates to anabsorbance of or change in polarization of the incident light.

The structure 110, or at least part thereof, may exhibit a forcedependent fluorescence in the sense that a higher or lower fluorescentlight intensity emerges from the structure 110 when a larger force isacting on it.

In case a force acting on a part of the structure is determined, whereinthe structure is a molecule, such as a DNA molecule, that part of thestructure may be a free segment of the molecule. Herein, a free segmentmay be understood to be a part of the molecule that is adjacentlyconnected only to one or more other parts of the same molecule(including the optically active entities), and not, for example,adjacently connected to a foreign structure. A foreign structure may bea bead to which the molecule is attached or may be another (DNA)molecule, for example one that is adjacently connected at a point wherethe two molecules are braided and/or fused. The method thus enables toobtain a force profile along a molecule, as opposed to one average forceacting on a molecule, or part of a molecule, fixed between two foreignobjects, for example two beads.

In one embodiment, the at least part of the structure comprises at leastone pair, preferably a plurality of pairs, of a donor fluorophore and anacceptor fluorophore. As known, an occurrence of energy transfer betweenthe donor and acceptor fluorophore, for example a Förster resonanceenergy transfer, may depend on a spatial distance between the donorfluorophore and the acceptor fluorophore. Further, the spatial distancebetween the donor fluorophore and the acceptor fluorophore may depend onthe force acting on the at least part of the structure. An increasingforce acting on the structure typically causes this distance toincrease. The donor fluorophore may emit light comprising a firstwavelength and the acceptor fluorophore may emit light comprising asecond wavelength different from the first wavelength. In thisembodiment, the light information may thus define a ratio betweenintensities of the first and second wavelength associated with the atleast part of the structure and the force may be determined based onthis ratio. In particular, this embodiment may comprise capturing awavelength spectrum of light from the structure 110 and determining theforce based on the spectrum. After all, the further apart the acceptorand donor are positioned, the lower the occurrence of energy transferbetween the two will be and the more prominent the first wavelength maybe present in the captured spectrum with respect to the secondwavelength. This embodiment may comprise, during the force measurement,predominantly exciting the donor fluorophore with an excitation lightsource and to a lesser extent exciting the acceptor fluorophore with anexcitation light source, e.g. not exciting the acceptor fluorophore. Inan example, the donor fluorophore is a green light emitting entity andthe acceptor fluorophore is a red light emitting entity. The pair ofdonor and acceptor then emit a spectrum that tends to contain highergreen light intensities with respect to red light intensities as theforce is increasing. In an example, the structure is a DNA moleculecomprising, e.g. along its length, alternately positioned donor andacceptor fluorophores.

FIG. 1B illustrates a method according to one embodiment. Optionallythis embodiment comprises the step S102. This step may serve to cause atleast part of the structure 110 to exhibit a force-dependent opticalactivity, such as a force-dependent fluorescence. This may namely enabledetermination of a force acting on the at least part of the structure inthe following steps S104-S106.

The force-dependent optical activity may relate to fluorescence as wellas to phosphorescence, luminescence, absorbance, polarization change, orany other optical phenomenon.

Step S102 comprises combining the at least part of the structure with afluid and optically active entities, for example in sample holder 108.Optically active may be understood to relate to at least one offluorescence, phosphorescence, luminescence, absorbance or any otheroptical phenomenon. The optically active entities in the fluid can bindto the at least part of the structure 110. A binding property of theoptically active entities in respect of the at least part of thestructure 110 depends on the force acting on the at least part of thestructure. The binding property in respect of the at least part of thestructure may relate to an off-rate and/or on-rate binding constant inrespect of the at least part of the structure and/or to a ratio betweenan off-rate and on-rate constant. The more optically active entities arebound to the at least part of the structure, the higher for example alight intensity may be of the light that emerges from the at least partof the structure.

Additionally or alternatively to the binding property, an opticalproperty of optically active entities, such as the above-described pairsof donor and acceptor fluorophores, bound to the at least part of thestructure 110 depends on the force acting on the at least part of thestructure 110. An optical property may relate to a quantum yield and/oran intensity and/or color and/or polarization of light emitted/absorbedby the optically active entities. The optical property may relate tocolor of light in the sense that the optical property relates to aspectrum, e.g. an emission or an absorbance spectrum of light. Thequantum yield may be defined for an optically active entity as thenumber of times a specific event, such as the emission of a photon,occurs per photon absorbed by the entity.

In one embodiment, the optically active entities comprise DNAintercalators, which may be understood to be planar molecules that bindreversibly between adjacent base-pairs of double-stranded (ds)DNA. Awide range of intercalator dyes are commercially available and theirDNA-binding properties have been characterized. In particular, theentities may be cyanine dyes. Then, DNA-binding affinity may vary by 2-4orders of magnitude over a force range of 0-60 pN.

In one embodiment, the fluorescent entities may comprise a labeledprotein and/or molecule that exhibits force-dependent optical activity,for example PICH.

In one embodiment, the entities when unbound to the at least part of thestructure exhibit a first optical activity and the entities when boundto the at least part of the structure exhibit a second optical activitythat is different from the first optical activity. In case the opticalactivity relates to fluorescence, the first and second optical activitymay relate to a first and second quantum yield respectively. Then, thesecond quantum yield may be 10-1000 times, preferably 500-1000 times,higher than the first quantum yield. Cyanine intercalator dyes, such asYO-PRO, YOYO-1, Sytox Orange and SYBR Gold, exhibit such enhancedfluorescence when intercalated. Enhanced fluorescence upon bindingadvantageously reduces background fluorescence because the amount oflight captured by the light sensitive system from non-bound fluorescententities is reduced with respect to the amount of light captured fromfluorescent entities bound to the structure 110.

In one embodiment, the entities exhibit an off-rate with respect to theat least part of the structure of at least, which off-rate is equal toor larger than a rate at which events of a process under scrutiny occur.In an example, the process concerns the separation of two strands of aDNA molecule by a helicase repeatedly performing a step of separating atleast one base pair at a time. Herein, the rate at which the eventoccurs may then relate to the number of times this separating step isperformed per unit of time. The equilibrium binding constant may bedefined as a ratio between an on-rate and the off-rate with respect tothe at least part of the structure. The off-rate may be tuned throughthe choice of ionic strength of the fluid. This embodimentadvantageously reduces perturbations of the structure and/or reducesperturbations of dynamic processes involving the structure, such asenzymes processing along DNA, because the entities only shortly bind tothe at least part of the structure. The binding time may advantageouslybe (tuned to be) shorter than the characteristic time of the processunder study in order to reduce perturbation of this process. Theoff-rate preferably is at least equal to, more preferably larger than,most preferably at least ten times larger than the rate at which theevents of the process of interest occur. For example, if a polymerasesteps at a rate of 100-1000 Hz, then it would be advantageous if theoff-rate is larger than 1000 to 10000 Hz to leave each step relativelyunperturbed. In another case, for example, where the averagetranslocation rate of this polymerase would be of interest, then theaverage intercalator coverage is preferably considered to correct forthe chance of encountering an intercalator. To illustrate, if apolymerase can on average travel ˜10 bases before it encounters anintercalator, then the off-rate should be equal to or larger than thestepping rate of the polymerase for the impact of the intercalator onthe rate to be less than ˜10%.

In one embodiment, a fractional coverage of the at least part of thestructure is lower than 50%. The structure may comprise binding sites atwhich the entities can bind. The fractional coverage may be defined as aratio between a number of binding sites at which an entity is bound anda total number of binding sites. In case the entities referred to areintercalators, a coverage rate of 50% under moderate concentration andtension conditions corresponds to one intercalator for every 4base-pairs of a DNA, because in such conditions, the footprint of anintercalator is 2 base pairs. Preferably, saturation is avoided becauseit may limit the dynamic range of fluorescence intensities, decrease thesensitivity of the force measurement, maximize any potentialperturbation, and the low average intercalator spacing at or nearsaturation could enhance self-quenching of the fluorescence signal,which complicates force measurements by introducing non-linearcontributions. This embodiment thus allows to measure the force over alarge force range, e.g. (˜1-65 pN), because non-linear effects due tosaturation and/or due to high coverage fractions are prevented.

Step S104 comprises controlling the light-sensitive system 102 todetermine light information based on light from the structure. Thelight-sensitive system 102 may be said to capture light when determiningthe light information and may be understood to comprise thelight-sensitive system storing or transmitting information, e.g. imagedata, based on light that is incident on at least a part, e.g. animaging plane, of the light-sensitive system 102.

The light information defines a light property value associated withsaid at least part of the structure 110. The light property value may beat least one of a light intensity value and a light color value and alight polarization value. A light intensity value associated with a partof the structure may indicate a radiant power of light from that part ofthe structure. A light color value may indicate a wavelength or awavelength range of the light from a part of the structure and/or alight spectrum and/or a ratio of intensities between multiple distinctwavelengths of light from a part of the structure and a lightpolarization value may indicate a plane in which the electric field ofthe light from the structure substantially oscillates.

The light information determined by the light-sensitive system 102 maynot comprise spatial information, for example may not define differentlight property values for respective parts of the structure 110. Inorder to perform such a bulk measurement, the light-sensitive system 102only requires a single pixel or large area detector. In one example, thestructure of interest 110 is a is a DNA containing structure (egpre-stressed DNA origami structure), at least partially positioned in afluorometer.

The light sensitive-system optionally comprises an imaging system thatmay comprise a plurality of pixels. Furthermore, the light informationmay comprise image data that may be spatially resolved, for example inthe sense that the image data define a plurality of light propertyvalues associated with respective parts of the structure. In such case,the image data may also be understood to define a single light propertyvalue for the collection of these parts in the sense that the image datadefine an average light property value, wherein the average lightproperty value may be an average of the plurality of light propertyvalues in the image data. Obtaining such an average light property valuemay comprise integrating said plurality of light property values.

Step S106 comprises, based on said light property value and on areference light property value, determining the force acting on the atleast part of the structure 110. The force may be determined in thesense that a magnitude and/or direction of the force is determined. Incase the magnitude of the force is measured, the magnitude may beunderstood to be the size of a force component acting in a specificdirection, such as a direction along a length of the structure 110.

In one embodiment the method comprises obtaining a parameter relating adifference of force magnitude to a difference in optical property valueand determining the force acting on the at least part of the structurebased on said parameter. In an example, a first force is acting on afirst part of the structure and a larger second force is acting on asecond part of the structure. In this example, the image data may definea first light intensity value for the first part and define a highersecond light intensity value for the second part. Based on the obtainedparameter, and based on the difference between the first and secondlight intensity value, a difference between the first and second forcemay be determined. In this example, the method comprises determining thesecond force in the sense that the method comprises quantifying how muchlarger the second force is with respect to the first force. Herein, thefirst force may be unknown.

FIG. 2A depicts an embodiment of the system wherein the system 200comprises a force application system 212 and FIG. 2B illustrates anembodiment comprising the step S200 of controlling the force applicationsystem 212 to apply a force to the at least part of the structure 210.In this disclosure, number indices in the figures that differ by hundredfolds indicate similar, e.g. identical, components. It should beappreciated that step S200 may be performed based on a force that waspreviously determined as indicated by the dashed line in FIG. 2B. Thisfeedback-loop enables to accurately apply forces to the at least part ofthe structure 212.

The force application system 212 may comprise a system for establishinga trap, such as an optical trap, such as a holographic optical trap,acoustical trap and electrical trap such as an Anti-BrownianElectrokinetic trap. It may also comprise a system for creatingacoustical standing waves in order to attract objects to the nodes ofthis standing wave and thereby apply forces on the objects. Thestructure 210 may be connected to at least one bead that sits in suchtrap established by system 212. In an example, the structure 210 isconnected to two trapped beads. In these cases, step S200 may comprisecontrolling the relative positions of the traps holding the beads. Itshould be understood that the force application system 212 may be anykind of system that can cause a force or a change of force acting on theat least part of the structure. An example of a force application systemwould thus be a manipulation system for deforming the at least part ofthe structure.

The structure 210 may be at least partially positioned in a flow cell208. The force application system 212 may then comprise the flow cell208 (not shown). Furthermore, step S200 may in such case comprisecontrolling at least one of a fluid flow and a solution of a fluid inthe flow cell 208, for example to control a drag force acting on the atleast part of the structure. The solution of the fluid may be controlledin the sense that the ionic strength of the solution is controlled.

In one embodiment, step S200 comprises controlling the force applicationsystem 212 to change the force acting on the at least part of thestructure 210. A change of the force may be achieved by a change ofposition of acoustical/optical/electrical traps relative to each other.A change of force may be achieved by varying the amplitude or wavelengthof an acoustic standing wave. A change of the force may be achieved bychanging at least one of a fluid flow and a solution of the fluid.

Step S204 may comprise controlling the light-sensitive system to capturelight from the structure while said force is changing. The lightinformation defines, for a plurality of time instances, a light propertyvalue associated with said at least part of the structure 210.Furthermore, step S206 may comprise, for each time instance, based onthe light property value, determining the force acting on the at leastpart of the structure. It should be appreciated that the step ofcontrolling the force application system to change the force acting onthe at least part of the structure may be performed based on an initialdetermination of the force in accordance with the methods describedherein as depicted by the dashed line.

With reference to FIG. 3 it is noted that in one embodiment the methodcomprises controlling the light-sensitive system to determine referencelight information based on reference light from a reference structurewhile a reference force is acting on at least a part of the referencestructure. In this embodiment, the at least part of the referencestructure exhibits a force-dependent optical activity. The referencelight information defines a reference light property value associatedwith said at least part of the reference structure.

Preferably, for the at least part of the reference structure thedependence of the optical activity on force is similar, e.g. the same,as for the at least part of the structure.

In this disclosure, reference forces may be applied to the structureusing a force application system as described herein. Optionally suchforce application system is calibrated which allows to apply knownforces to the reference structure. In an example, the structure istethered to a bead, which bead may be trapped, for example in an opticalor acoustical trap.

The application of reference forces does not require the use ofcalibrated traps. In one example, a reference force of zero is applied,which may be established by removing or minimizing the mechanicalperturbation to the structure. In another example, the embodimentcomprises determining the reference force based on one or more observedevents associated with one or more forces. To illustrate, in case thestructure is a DNA molecule it is known that, in the presence ofintercalators, the onset of DNA overstretching typically occurs between65 and 70 pN. Hence, when the imaging system captures the referencefluorescent light from the structure at the moment the DNA starts tooverstretch, the reference light property value as defined by the lightinformation can be associated with 65-70 pN, which allows calibrationfor measuring absolute forces. In yet another example, the opticallyactive entities may become active when a force acting on the at leastpart of the structure exceeds a threshold force. Then, the determinedlight property value at the moment the optically active entities becomeactive can be associated with the threshold force.

FIG. 3A visualizes reference light information, in particular referenceimage data 316, that defines for a plurality of time instances t1-t10 anaverage light intensity value for a single DNA molecule 310. At a timet1 a reference force F1 of approximately 7 pN is applied to the DNAmolecule 310, in this example the reference structure, by controllingthe relative positions of two calibrated optical traps 322 a and 322 bholding beads. The vertical axis of the graph 3B shows force values andthe horizontal axis shows light property values, in this casefluorescence light intensity values, that are associated with the DNAmolecule 310. An imaging system has thus determined, based on referencelight from the DNA molecule 310, which reference light was capturedwhile the reference force F1 was applied to the DNA molecule 310,reference image data 316 defining a reference property value I1.Therefore, graph 3B shows point (I1, F1). The processor may store thereference property value I1 in association with reference force F1.

If both the above-described parameter has been obtained as well as thereference light property value I1 in association with a known referenceforce F1, then the absolute force F2 acting on the at least part of thestructure may be determined based on I2. Herein, the following relationmay be used

$\begin{matrix}{{F\; 2} = {{{\phi ln}\left( \frac{I_{2}}{I_{1}} \right)} - {F\; 1.}}} & (1)\end{matrix}$

I2 denotes the light property value as defined by the light informationdetermined by the light-sensitive system and ϕ said parameter.

In case the above-described parameter has been obtained as well as thereference light property value I1, wherein the reference light propertyvalue is associated with an unknown reference force, then, a forcedifference between the unknown reference force and the force acting onthe at least part of the structure may be determined, for example basedon the relation

$\begin{matrix}{{\Delta\; F} = {{{\phi ln}\left( \frac{I_{2}}{I_{1}} \right)}.}} & (2)\end{matrix}$

It should be appreciated that a difference may relate to a subtractionand/or to a ratio. To illustrate, the parameter may be the parameter ϕas per above, which relates a difference in force to a difference inlight intensity value, wherein the force difference relates to asubtraction and the intensity difference is expressed as a ratio betweenI₂ and I₁. A force increase of ΔF=F2−F1 causes an intensity increases bya factor of e^(ΔF/ϕ).

Yet a further embodiment comprises controlling the light sensitivesystem to determine second reference light information based on secondreference light from a second reference structure while a secondreference force is acting on at least a part of the second referencestructure. The second reference light information defines a secondreference light property value associated with said at least part of thesecond reference structure. This embodiment further comprisesdetermining the force acting on the at least part of the structure basedon the second reference light property value. In the example shown inFIG. 3, the reference structure 310 is the same structure as the secondreference structure. FIG. 3B shows that at a time t4 a second referenceforce F2 of approximately 25 pN was applied to the DNA molecule 310.Furthermore, the second reference light information defines a secondreference light property value of I2 for the second reference force F2.

Based on the reference light property value I1 associated with thereference force F1 and the second reference light property value I2associated with the second reference force F2, the parameter relatingthe parameter relating a difference of force magnitude to a differencein optical property value may be determined. This embodiment thusenables to independently determine this parameter.

In particular, FIG. 3A presents snapshots of fluorescence imagesrecorded for a dsDNA molecule (˜48.5 kb) under increasing tension in thepresence of the cyanine dye YO-PRO (YO) (10 nM). A clear increase influorescence intensity is observed as the applied force is raised. Thistrend is quantified in FIG. 3B for cyanine dye YO at 10 nM concentrationand for Sytox Orange (SxO) for 6 and for 20 nM concentration. In orderto accurately describe the relationship between intercalatorfluorescence and dsDNA tension, it is noted that the equilibrium bindingconstant (K) of an intercalator depends on the force (F) applied to theDNA molecule:

$\begin{matrix}{{K(F)} = {K_{0}e^{\frac{F}{\phi}}}} & (3)\end{matrix}$

Here, K₀ is the binding affinity of the intercalator for dsDNA at 0 pNand ϕ is a characteristic force equal to k_(B)T/ΔX_(eq), where ΔX_(eq)represents the equilibrium average elongation per base-pair due to thepresence of an intercalator dye. The equilibrium average elongation perbase-pair due to the presence of an intercalator dye formono-intercalators is approximately 0.34 nanometer (corresponding to 12pN) and for bis-intercalators approximately 0.68 nanometer(corresponding to 6 pN). Equation 3 results from the fact that anapplied force lowers the net free energy cost associated with theincreased DNA extension upon intercalation. Further, it is known thatthe equilibrium binding constant can be related to both theconcentration of intercalator [I] and the fractional dye coverage ϑ asfollows:

$\begin{matrix}{K = {\frac{1}{n\lbrack I\rbrack}\frac{\vartheta}{\left( {1 - \vartheta} \right)}}} & (4)\end{matrix}$

where n is the footprint of the intercalator in base-pairs, and ϑ variesbetween 0 and 1. Whereas equation (4) is based on a multiligand bindingisotherm, other binding isotherms may be used that are appropriate forcertain substrate—ligand interactions. One such isotherm that is oftenused for DNA-binding ligands is the McGhee von Hippel isotherm as per J.D. McGhee and P. H. von Hippel, J. Mol. Biol. (1976) 103, 679. Inaddition, it has also been established that: (i) the elongation of dsDNAdue to the binding of cyanine dye intercalators scales linearly with thefluorescence intensity; and (ii) dsDNA elongation is directlyproportional to d. Extrapolating the above laws it can be derived thatthe total (background-corrected) intercalator fluorescence intensity(I_(F)) is related to the applied force on dsDNA by the followingexpression:

$\begin{matrix}{F = {- {{\phi ln}\left\lbrack {\frac{B}{I_{F}}\left( {I_{\max} - I_{F}} \right)} \right\rbrack}}} & (5)\end{matrix}$

I_(max) (the maximum background-corrected fluorescence intensity atsaturated coverage) and B are parameters defined as follows.

$\begin{matrix}{I_{\max} = \frac{I_{n}N_{bp}}{n}} & (6) \\{and} & \; \\{B = {{nK}_{0}\lbrack I\rbrack}} & (7)\end{matrix}$

wherein I_(n) is the background-corrected fluorescence intensity of asingle intercalator dye, N_(bp) is the number of base-pairs in the DNAmolecule.

Fitting the data in FIG. 3B to Eqn. 5 (solid lines) provides anexcellent match over the full range of forces considered (0-60 pN).Hence, the step of determining the force acting on the at least part ofthe structure may be performed based on a relation defined by Equation5.

ϕ may be determined by plotting the elongation in dsDNA length due tothe binding of intercalators against the number of intercalatedmolecules. A linear fit to this plot yields Δx_(eq), from which ϕ canthen be calculated.

In order to extract I_(max) and B, two alternative methods may beemployed. In the first method, the assertion is made that I_(max) and Bcan be expressed via the following two equations:

$\begin{matrix}{I_{\max} = {I_{F}\left( {1 + \frac{e^{{- F}/\phi}}{B}} \right)}} & (8) \\{B = \frac{I_{F}e^{{- F}/\phi}}{I_{\max} - I_{F}}} & (9)\end{matrix}$

Assuming two reference force values are known, denoted here as F₁ and F₂(with corresponding fluorescence intensity values I₁ and I₂), thefollowing may be derived:

$\begin{matrix}{I_{\max} = {{I_{F\; 1}\left( {1 - \frac{e^{{- F_{1}}/\phi}}{e^{{- F_{2}}/\phi}}} \right)}\left( {1 - {\frac{I_{F\; 1}}{I_{F\; 2}}\frac{e^{{- F_{1}}/\phi}}{e^{{- F_{2}}/\phi}}}} \right)^{- 1}}} & (10)\end{matrix}$

By inserting the known reference forces (along with their measuredfluorescence intensities) into Eqn. 10, I_(max) can be calculated. Themagnitude of B can then be determined by inserting this value of I_(max)into Eqn. 9. The reference forces may be: (i) 0 pN, which can beestablished by removing or minimizing the mechanical perturbation to thedsDNA; and (ii) the onset of dsDNA overstretching, which typicallyoccurs at 70±3 pN in the presence of intercalators.

In the second method, Imax and B are determined from a fit of Eqn. 5using either: (i) two reference force values (along with theircorresponding fluorescence intensities) at a constant intercalatorconcentration, or (ii) two reference intercalator concentrations (alongwith the associated fluorescence intensities) at a constant known force.

The fluorescence intensity increases mono-exponentially with appliedforce when the dye coverage is far from saturation (i.e. at low forceranges and/or at lower dye concentrations). ϑ is then namelyproportional to K (see equation 1). On the basis of this, any change inapplied force (ΔF=F₂−F₁) can be determined simply by using the followingrelation:

$\begin{matrix}{{\Delta\; F} = {{\phi ln}\left( \frac{I_{2}}{I_{1}} \right)}} & (2)\end{matrix}$

where I_(i) is the background-corrected fluorescence intensity at F_(i).In this case, the absolute value of an unknown force (say F₂) can thenbe calculated by comparing the fluorescence intensity at this force withthat at a known reference force (F₁). A useful reference force herecould be 0 pN. The dashed lines in FIG. 3B confirm that, indeed, amono-exponential fit describes the measured data well under low coverageconditions (i.e. up to ˜45 pN at this dye concentration).

One embodiment comprises determining that the intercalator coverage isfar from saturation. This embodiment comprises controlling a forceapplication system to apply a known force to the structure, e.g. DNA,while the fluorescence intensity is measured at increasing intercalatorconcentrations, [I]. A plot of fluorescence intensity versus [I] will belinear until levelling off at higher [I] due to saturation (Eqn. 5). Theconcentration range over which intercalated dsDNA is out of saturationcan then be estimated from the linear region of this curve. Note thatsaturation will occur at lower intercalator concentrations as theapplied force is increased. It is therefore preferable to perform thissaturation test at the highest force expected during experiments. A moreapproximate, but simpler, approach is to measure the fluorescenceintensity at a plurality of intercalator concentrations whilemaintaining the structure at a fixed force. If the fluorescenceintensity increases by the same factor as does the intercalatorconcentration, it can be assumed, to a first approximation, that the dyecoverage is far from saturation.

The force resolution of this method depends on the signal-to-noise ratioassociated with the fluorescence images. When for example thefluorescence signal from YO (10 nM) for each pixel (130 nm) of animaging system is captured over a time interval between 3 and 17seconds, at forces between 60 and 6 pN, a force resolution may beachieved in the range of 1-3 pN.

With reference to FIG. 4 it is noted that in one embodiment, thelight-sensitive system comprises an imaging system. In this embodiment,the light information comprises image data, wherein the image datadefine, for a plurality of time instances t1-t8, a set of light propertyvalues associated with respective parts of the structure. Thisembodiment comprises the step of, for each part, based on its associatedlight property value, determining a force acting on the part of thestructure.

FIG. 4 shows image data 416 that may be obtained in case thelight-sensitive system comprises an imaging system and the lightinformation comprises image data. Herein, the structure 410 is at leastpartially positioned in a flow cell and the force acting on at leastpart of the structure comprises a drag force caused by a fluid flow.FIG. 4A shows a DNA molecule 410 that is tethered on one end to anoptically-trapped bead 422. The DNA molecule 410 is stretched by ahydrodynamic flow indicated by the arrow. The image data 416 define aset of light property values associated with respective parts of thestructure 410. In FIG. 4A, the image data 416 define a first lightproperty value, e.g. an average recorded intensity, associated with part418 of the structure 410 and define a second light property valueassociated with part 420 of the structure.

A maximum dimension of the respective parts for example is smaller than5000 nanometer, preferably smaller than 2000 nanometer, more preferablysmaller than 500 nanometer.

In principle, the spatial resolution with which forces can be resolvedis the spatial resolution of the microscopy technique applied. Thesmallest part than can be resolved using standard optical microscopy isapproximately ½ times the wavelength of excitation/detection light, i.e.approximately 200-300 nm. For example, with STED the smallest part canin principle be arbitrary small but in practice parts of approximately20 nm can be resolved. Using localization techniques small parts ofapproximately 5 nm can be resolved.

The imaging system may determine the image data in parallel. In anexample, the imaging system comprises a plurality of pixels. Each pixelmay receive light from a respective part of the structure. A pixel maythus perform the step of determining light information based on lightfrom an associated part of the structure. The pixels may simultaneouslyperform this step. Thus the imaging system may image the respectiveparts of the structure at once.

It should be appreciated that the image data 416 may represent an imageof the at least part of the structure 410 and comprises a set of imagepixel values associated with respective parts of the structure.Furthermore, the method may comprise determining one or more subsets ofone or more image pixel values, for example the image pixel values ofthe image pixels within area 418 indicated in the image. Each subsetdefines a region of interest (ROI) 418 in the image. For each ROI, theforce acting on a part of the structure represented by the ROI 418 maybe determined based on the image pixel values defining the ROI 418.

FIG. 4B shows that, in case the imaging system is controlled to capturelight from the at least part of the structure 410 while the force actingon the at least part of the structure is changing, the image data 416may define, for a plurality of time instances t1-t8, a set of lightproperty values associated with respective parts of the structure. Thisallows to monitor changing forces acting on a plurality of parts of thestructures.

In particular, FIG. 4 shows experiments wherein a lambda-DNA molecule,tethered on one end to an optically-trapped bead (1.84 micrometerdiameter), was stretched via hydrodynamic flow (arrow) in the presenceof intercalator dye. FIG. 4A displays sample fluorescence imagesobtained as the DNA is stretched using different flow speeds in thepresence of SxO (20 nM). FIG. 4B shows tension along the length of theDNA molecule shown in FIG. 4A, in this example as a function of flowspeed. The tension over the different segments of the DNA was derivedusing Eqn. 5. The flow speed was calculated using Stokes' law. FIG. 4Cshows a comparison of the maximum drag force, near the tethered end ofthe dsDNA molecule (calculated using Eqn. 5), as a function of flowspeed for two different dyes: YO (10 nM) and SxO (6 nM, 20 nM). Datawere obtained in a buffer containing 20 mM HEPES pH 7.5, 100/150 mMNaCl, 2/10 mM MgCl2, 0.025 casein and 0.05% Pluronics F127 for YO/SxOstudies, respectively.

FIG. 5A illustrates an experimental scheme according to one embodiment,wherein a dsDNA molecule (˜8.6 kb) is tethered between the surface of aflow-cell and a bead of 1.84 micrometer diameter. The DNA molecule isstretched using hydrodynamic flow. FIG. 5B shows sample fluorescenceimages of a flow-stretched dsDNA molecule in the presence of SxO (20 nM)as the shear flow is increased (frames 1-9). FIG. 5C shows an averageDNA drag force as a function of shear flow. The drag force wascalculated using Eqn. 5, while the shear flow was tuned through thepressure applied to the reservoir containing the intercalator solution.Note that black circular data points correspond to the snapshots in FIG.5B, while triangular data points are derived from fluorescence images ofa second surface-tethered DNA molecule. Data were obtained in a buffercontaining 20 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.05% caseinand 0.1% Pluronics F127.

With reference to FIG. 6, it is noted that in one embodiment, thestructure comprises at least one DNA molecule that is connected to (e.g.through covalent bonds, electrostatic or other attractive interactionsor topologically by e.g. braiding) a second molecule, for example asecond DNA molecule. More complex tension patterns and mechanicallyordered structures (e.g. chromosomes) can often arise in two-dimensionalor three-dimensional molecular architectures, as commonly encountered inliving systems. One such example involves the entanglement of multipleDNA molecules or compacted chromosomes. The study of entwined DNA is ofconsiderable interest from both a physical and biological perspective.It has recently been discovered, for instance, that stretched andentwined dsDNA tracts are generated regularly in vivo during chromosomesegregation through the formation of so-called ultra-fine DNA bridges.This embodiment advantageously enables to measure how forces arepartitioned within multi-component DNA complexes.

FIG. 6 illustrates that the methods disclosed herein may be used tomeasure the force response of two entwined dsDNA molecules as they areplaced under tensile strain. To generate entangled DNA, a 4-way opticaltrap may be used to manipulate two dsDNA molecules, each one heldbetween a set of two optically-trapped beads. By translating the beadsin 3-dimensions, one dsDNA molecule may be wrapped once around the otherto create an entwined dual dsDNA structure consisting of four ‘arms’.Each arm may be tethered to a different bead (numbered 1-4), asillustrated in FIG. 6A. The entwined DNA assembly may be incubated inSxO (20 nM) and the fluorescence within a region of interest (ROI) alongeach arm of the DNA structure may be imaged. Following this, tensilestrain may be applied to the entwined DNA structure by first increasing,and then decreasing, the distance between bead 41 and bead 42 (delta d),shown schematically in FIG. 6C. From the change in fluorescenceintensity within ROI 41, Eqn. 4 may be used to measure the reduction intension within arm #1 as Δd is decreased (following its initialextension). The results of this are shown in FIG. 4C (circular datapoints). Note that the absolute force was calibrated with reference tothe thermal fluctuations of bead 41 in the optical trap, determinedusing back-focal plane interferometry (shown as a line in FIG. 4c ).Together, these data (which show good agreement) reveal that an abrupt,and unexpected, drop in force occurs as Δd is decreased.

In order to understand the nature of this sudden decrease in force,intercalator fluorescence alone may be used to extract the change inforce on all four dsDNA arms. The lower panel of FIG. 4C compares thefluorescence images recorded before (i), during (ii) and after (iii) theabrupt change in force identified above. FIG. 4D quantifies the changein tension within each arm of the entwined DNA complex between frames(i) and (iii), using Eqn. 4 and based on the corresponding change influorescence intensity within ROI #1-4, respectively. This analysisreveals that a sudden reduction in force of ˜15 pN in fact occurs alongboth arms #1 and #3, while an increase in force of ˜5-10 pN is detectedalong both arms #2 and #4. Thus, using intercalator fluorescence as aforce probe, it may be confirmed that a substantial and abruptrearrangement in tension occurs along each ‘diagonal’ component of theentangled DNA complex as Δd is decreased. An additional—andcomplementary—advantage of the use of cyanine fluorescence is that itcan easily correlate rearrangements in force with changes in DNAgeometry. With this in mind, FIG. 6E compares the location of theintersection point of the two entwined dsDNA molecules before and afterthe force jump identified in FIGS. 6C and 6D. From this, it may bedetermined that, at the moment of the force rearrangement, theintersection point shifts by ˜500±100 nm.

This example highlights how intercalator fluorescence can be exploitedto quantify, in remarkable detail, the redistribution of local forceswithin complex DNA architectures. For instance, using the methods asdisclosed herein, also stick-slip dynamics when sliding one entwined DNAmolecule through another may be detected and measured.

While the light-sensitive system is capturing light from the structure,the structure may be positioned in a buffer containing 20 mM HEPES pH7.5, 100/150 mM NaCl, 2/10 mM MgCl2, 0.02-0.05% casein and 0.05-0.1%Pluronics F127 for YO/SxO studies, respectively. Alternatively, thestructure may be positioned in a buffer of 20 mM Tris-HCl pH 7.5 and 50mM NaCl. Stock solutions of intercalators (YO and SxO) may be preparedin dimethylsulphoxide (DMSO) and stored at −20 degrees Celsius. Prior toperforming force measurements, intercalator solutions may be dilutedwith buffer (with DMSO constituting less than 2% v/v). The structure maybe bacteriophage lambda DNA (˜48.5 kb). In surface-based flow-stretchassays, a shorter DNA construct may be employed (derived from alinearized PKYB-1 plasmid, ˜8.6 kb).

The light-sensitive system may be part of an inverted wide-fieldfluorescence microscopy. Furthermore, dual-trap optical tweezers may beused. The force measurements may be performed in a multi-channel laminarflow-cell where lambda-DNA is tethered between two microspheres (ofdiameter 1.84 micrometer) in situ. Such a flow-cell allows this dumbbellconstruct to be exchanged rapidly between channels containing differentbuffer conditions or intercalator dyes. Forces applied to the lambda-DNA(via displacement of a tethered microsphere) may be measuredindependently via back-focal plane interferometry of the condenser toplens using a position-sensitive detector. Fluorescence from DNAintercalators may be induced with either a 491 nm excitation laser (YO)or a 532 nm excitation source (SxO). The resulting fluorescence may beimaged using an EMCCD camera as light sensitive-system.

For a surface-based DNA flow-stretch assay, DNA molecules (˜8.6 kb) maybe tethered between a microsphere and a glass surface using a protocolmodified from a standard procedure. A custom-made glass flow-cell withan interior channel of dimensions 8000×2000×100 μm may be connected, viaa tubing system, to a reservoir inside a pressure box. The surface ofthe flow-cell may first be coated with Anti-Digoxigenin (ADig)antibodies in PBS (0.02 mg/ml) for 30 minutes. After rinsing with PBS,the surface may be incubated sequentially with BSA (2 mg/ml), thenPluronics F127 (5 mg/ml), each in PBS. Surface-tethered DNA may begenerated by incubating the DNA (10 pM in PBS, pre-labelled withBiotin/Digoxigenin on opposite ends) in the flow-cell for 20 minutes,before blocking residual ADigs with a solution of digoxigenylated caseinin PBS (0.05 mg/ml) for 30 minutes. Following this, the surface-tetheredDNA may be incubated with 25 microliter of a 0.1% suspension ofstreptavidin-coated microspheres (1.84 μm diameter) in PBS (containing0.02 of both casein and Tween 20) for 30 minutes. Untetheredmicrospheres may then be flushed out of the flow-cell with themeasurement buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.05%casein and 0.1% Pluronics F127). Note that the hydrodynamic flow rate islower near the surface of a flow-cell than in the centre due to themeniscus effect; when flow-stretching a surface-tethered DNA, the forceapplied to the DNA will therefore be lower near the tethering point. Forthis reason, fluorescence intensity may be measured in a region ofinterest away from the surface-tethered end of the DNA (where themolecule can be considered near parallel to the surface).

For combined 4-way optical trapping and fluorescence microscopy, theoutput of a 1064 nm fibre laser may be split into four paths which maythen be used to generate four optical traps within an invertedmicroscope. The methods for force measurements may be performed in amulti-channel flow-cell where two dumbbell constructs are constructed insitu, each composed of a lambda-DNA molecule tethered between twomicrospheres (of diameter 3.5 micrometer). Entwinement of two DNAmolecules may be achieved by displacing two of the four microspheres in3-dimensions.

The microscope of which the light-sensitive system may be part may be acustom-built inverted microscope that combines wide-field fluorescencemicroscopy and dual-trap optical tweezers. A 1,064-nm fibre laser(YLR-10-LP, 10 W, IPG Photonics) and a water-immersion objective (PlanApo 60, numerical aperture 1.2, Nikon) may be used to create twoorthogonally polarized optical traps. The trap separation may becontrolled using a piezo mirror (Nano-MTA2X Aluminium, Mad City Labs)for beam-steering one trap. Force measurements using the traps may beperformed by back-focal plane interferometry of the condenser toplens (P1.40 OIL S1 11551004, Leica) using a position-sensitive detector(DL100-7PCBA3, Pacific Silicon Sensor). Fluorescence microscopy may beachieved by imaging the stained DNA on an EMCCD camera (iXON

897E, Andor Technology). A 491-nm excitation laser (Cobolt Calypso 50 mWCW), and HQ540/80 m bandpass filter (Chroma Technology), may be used forimaging of YO-PRO-1, YOYO-1, SYBR Gold and SYTOX Green, and a 532-nmexcitation laser (Cobolt Samba 50 mW CW) and FF01-580/60 bandpass filter(Semrock Inc.) may be used for imaging of SYTOX Orange and POPO-3.

The sample holder and/or the force application system may comprise amultichannel laminar flow cell mounted on an automated XY-stage(MS-2000, Applied Scientific Instrumentation), which allows rapid insitu construction and characterization of dumbbell constructs, andfacilitates swift and complete transfer of tethered DNA betweendifferent flow channels. The flow cell and microspheres may beilluminated by a 450-nm light emitting diode (Roithner LasertechnikGmbH) and imaged in transmission on a CMOS camera (DCC1545M, Thorlabs).

Intercalators are known to nonspecifically bind to glass surfaces andmicrofluidic tubing with high affinity. Such surface adsorption reducesthe dye concentration in solution right after introducing dyes to amicrofluidic system or after increasing the dye concentration. Surfacedesorption, on the other hand, increases the dye concentration whenswitching to a lower dye concentration. In order to ensure that duringmeasurements a constant and well-defined dye concentration is present,the microfluidic system may be equilibrated using the dye-containingbuffer. In equilibrium, surface adsorption equals dye desorption.Equilibration may be accomplished by thorough flushing with a largevolume of intercalator. Equilibration of the dye concentration may beconfirmed by monitoring changes in DNA elongation. A channel may beconsidered to be equilibrated in case the DNA elongation remainsunaltered after additional flushing and remained constant over repeatedmeasurements. The channel equilibration time may depend on theintercalator used. For example, YOYO (which exhibits a relatively strongglass adsorption properties) required at least 24 h of flushing forequilibration.

FIG. 7A illustrates that, in one embodiment, the structure 710 isconnected to another structure 726, such as a cell, protein, virusparticle, DNA or RNA molecule or any other structure. In this case theforce acting on at least part of the structure 710 is exerted by theother structure 726 on the structure. Optionally the method comprisesconnecting the structure 710 and the other structure 726. The structure710 may also be connected to yet another structure 724 such as a surface724, e.g. a functionalized surface. The structure 710 may thus functionas a connector between structure 724 and structure 726, e.g. as a DNAlinker molecule. The connection between the structure and anotherstructure may be physical, for example may comprise a covalent bond.Additionally or alternatively, this connection may be topological, whichmay be the case when both the structure 710 and the other structure 726are entangled, for example as two linked ring shaped structures.

FIG. 7B illustrates that, in one embodiment, the method comprisescontrolling a force application system 712 to apply a force to the otherstructure 726.

As shown in the figure, this force need not be applied to the otherstructure 726 through the structure 710. In the example shown, the otherstructure 726 is a protein that is linked by the structure 710 to asurface 724. The protein 726 may additionally be connected to a trappedbead 722 and the position of the trap 728 holding the bead 722 may becontrollable by the force application system 712 as described above.This embodiment allows to investigate the protein's mechanical responseto the force applied to it. Herein, the mechanical response may thus beassessed by determining the force that the protein 726 applies to thestructure 710 in response to the force applied by the force applicationsystem 712 to the other structure 726.

FIG. 8 depicts a block diagram illustrating an exemplary data processingsystem that may be used in a computing system as described withreference to FIG. 1A.

As shown in FIG. 8, the data processing system 800 may include at leastone processor 802 coupled to memory elements 804 through a system bus806. As such, the data processing system may store program code withinmemory elements 804. Further, the processor 802 may execute the programcode accessed from the memory elements 804 via a system bus 806. In oneaspect, the data processing system may be implemented as a computer thatis suitable for storing and/or executing program code. It should beappreciated, however, that the data processing system 800 may beimplemented in the form of any system including a processor and a memorythat is capable of performing the functions described within thisspecification.

The memory elements 804 may include one or more physical memory devicessuch as, for example, local memory 808 and one or more bulk storagedevices 810. The local memory may refer to random access memory or othernon-persistent memory device(s) generally used during actual executionof the program code. A bulk storage device may be implemented as a harddrive or other persistent data storage device. The processing system 800may also include one or more cache memories (not shown) that providetemporary storage of at least some program code in order to reduce thenumber of times program code must be retrieved from the bulk storagedevice 810 during execution.

Input/output (I/O) devices depicted as an input device 812 and an outputdevice 814 optionally can be coupled to the data processing system.Examples of input devices may include, but are not limited to, akeyboard, a pointing device such as a mouse, or the like. Examples ofoutput devices may include, but are not limited to, a monitor or adisplay, speakers, or the like. Input and/or output devices may becoupled to the data processing system either directly or throughintervening I/O controllers.

In an embodiment, the input and the output devices may be implemented asa combined input/output device (illustrated in FIG. 8 with a dashed linesurrounding the input device 812 and the output device 814). An exampleof such a combined device is a touch sensitive display, also sometimesreferred to as a “touch screen display” or simply “touch screen”. Insuch an embodiment, input to the device may be provided by a movement ofa physical object, such as e.g. a stylus or a finger of a user, on ornear the touch screen display.

A network adapter 816 may also be coupled to the data processing systemto enable it to become coupled to other systems, computer systems,remote network devices, and/or remote storage devices throughintervening private or public networks. The network adapter may comprisea data receiver for receiving data that is transmitted by said systems,devices and/or networks to the data processing system 800, and a datatransmitter for transmitting data from the data processing system 800 tosaid systems, devices and/or networks. Modems, cable modems, andEthernet cards are examples of different types of network adapter thatmay be used with the data processing system 800.

As pictured in FIG. 8, the memory elements 804 may store an application818. In various embodiments, the application 818 may be stored in thelocal memory 808, the one or more bulk storage devices 810, or apartfrom the local memory and the bulk storage devices. It should beappreciated that the data processing system 800 may further execute anoperating system (not shown in FIG. 8) that can facilitate execution ofthe application 818. The application 818, being implemented in the formof executable program code, can be executed by the data processingsystem 800, e.g., by the processor 802. Responsive to executing theapplication, the data processing system 800 may be configured to performone or more operations or method steps described herein.

In one aspect of the present invention, the data processing system 800may represent a controller as described herein.

Various embodiments of the invention may be implemented as a programproduct for use with a computer system, where the program(s) of theprogram product define functions of the embodiments (including themethods described herein). In one embodiment, the program(s) can becontained on a variety of non-transitory computer-readable storagemedia, where, as used herein, the expression “non-transitory computerreadable storage media” comprises all computer-readable media, with thesole exception being a transitory, propagating signal. In anotherembodiment, the program(s) can be contained on a variety of transitorycomputer-readable storage media. Illustrative computer-readable storagemedia include, but are not limited to: (i) non-writable storage media(e.g., read-only memory devices within a computer such as CD-ROM disksreadable by a CD-ROM drive, ROM chips or any type of solid-statenon-volatile semiconductor memory) on which information is permanentlystored; and (ii) writable storage media (e.g., flash memory, floppydisks within a diskette drive or hard-disk drive or any type ofsolid-state random-access semiconductor memory) on which alterableinformation is stored. The computer program may be run on the processor802 described herein.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed. Thedescription of embodiments of the present invention has been presentedfor purposes of illustration, but is not intended to be exhaustive orlimited to the implementations in the form disclosed. Many modificationsand variations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the present invention.The embodiments were chosen and described in order to best explain theprinciples and some practical applications of the present invention, andto enable others of ordinary skill in the art to understand the presentinvention for various embodiments with various modifications as aresuited to the particular use contemplated.

The invention claimed is:
 1. A computer-implemented method fordetermining a force acting on at least part of a structure, for example,a biological structure, such as a DNA molecule, the method comprisingsteps of: controlling a light-sensitive system, e.g. a light sensitivesystem of a microscope, to determine light information based on lightfrom the structure, the light being incident on at least a part of thelight sensitive system, wherein the at least part of the structurecomprises one or more optically active entities, such as DNAintercalator molecules, at least one of an optical activity of theentities and a quantity of the entities depends on the force acting onthe at least part of the structure and the light information defines alight property value associated with said at least part of thestructure; and determining the force acting on the at least part of thestructure on the basis of said light property value.
 2. The methodaccording to claim 1, further comprising a step of controlling a forceapplication system to apply a further force to the at least part of thestructure on the basis of the force determined in the determining step.3. The method according to claim 1, wherein the light-sensitive systemcomprises an imaging system and the light information comprises imagedata defining a set of light property values associated with respectiveplural structures comprising said structure, the method comprising foreach structure of the plural structures, a step of determining a forceacting on the structure based on its associated light property value. 4.The method according to claim 1, wherein the light-sensitive systemcomprises an imaging system and wherein the light information comprisesimage data representing an image of the at least part of the structureand comprising a set of image pixel values associated with respectiveparts of the structure, wherein said step of determining the forcecomprises determining one or more subsets of one or more image pixelvalues, each subset defining a region of interest (ROI) in the image;and for each ROI, determining the force acting on a part of thestructure represented by the ROI based on the image pixel valuesdefining the ROI.
 5. The method according to claim 1, further comprisinga step of obtaining a parameter relating a difference of force magnitudeto a difference in optical property value, and determining the forceacting on the at least part of the structure based on said parameter. 6.The method according to claim 1, further comprising a step of:controlling the light-sensitive system to determine reference lightinformation based on reference light from a reference structure while areference force is acting on at least a part of the reference structure,the reference light being incident on at least a part of thelight-sensitive system, wherein the at least part of the referencestructure comprises one or more optically active reference entities andwherein at least one of an optical activity of the reference entitiesand a quantity of the reference entities depends on the reference forceacting on the at least part of the reference structure and wherein thereference light information defines the reference light property valuethat is associated with said at least part of the reference structure.7. The method according to claim 6, further comprising controlling thelight sensitive system to determine second reference light informationbased on second reference light from a second reference structure whilea second reference force is acting on at least a part of the secondreference structure, the second reference light being incident on atleast a part of the light-sensitive system, wherein the at least part ofthe second reference structure comprises one or more optically activesecond reference entities and wherein at least one of an opticalactivity of the second reference entities and a quantity of the secondreference entities depends on the second reference force acting on theat least part of the second reference structure and wherein the secondreference light information defines a second reference light propertyvalue associated with said at least part of the second referencestructure, and wherein the method comprises determining the force actingon the at least part of the structure based on the second referencelight property value.
 8. The method according to claim 1, wherein thestructure is at least partially positioned in a fluid comprisingoptically active entities, and wherein a binding property of theoptically active entities in respect of the at least part of thestructure depends on the force acting on the at least part of thestructure.
 9. The method according to claim 8, wherein while thereference light is incident on at least a part of the light-sensitivesystem, a first concentration of optically active entities is present inthe fluid; the method further comprising controlling the light-sensitivesystem to determine second reference light information based on secondreference light from the reference structure while the reference forceis acting on at least a part of the reference structure, the secondreference light being incident on at least a part of the light-sensitivesystem, wherein while the second reference light is incident on the atleast a part of the light-sensitive system, a second concentration ofoptically active entities different from the first concentration ispresent in the fluid, and wherein the second reference light informationdefines a second reference light property value associated with said atleast part of the reference structure, and wherein the method comprisesdetermining the force acting on the at least part of the structure basedon the second reference light property value.
 10. The method of claim 8,wherein the structure is at least partially positioned in a sampleholder containing the fluid.
 11. The method according to claim 1,wherein the structure is connected to another structure, and wherein theforce acting on at least part of the structure is exerted by the otherstructure on the structure.
 12. The method according to claim 1, whereinthe structure is at least partially positioned in a flow cell, andwherein the force acting on at least part of the structure comprises adrag force caused by a fluid flow.
 13. The method according to claim 1,wherein the optically active entities comprise at least one pair of adonor fluorophore and an acceptor fluorophore, the at least one pairexhibiting an emission spectrum that depends on the force acting on thestructure.
 14. The method of claim 13, wherein the optically activeentities comprise a plurality of pairs of a donor fluorophore and anacceptor fluorophore, each said pair exhibiting an emission spectrumthat depends on the force acting on the structure.
 15. A computerprogram comprising instructions which, when the program is executed by acomputer, cause the computer to carry out one or more of the steps ofthe method as defined in claim
 1. 16. A method for causing at least partof a structure to exhibit a force-dependent optical activity forenabling determination of a force acting on the at least part of thestructure in accordance with claim 1, the method comprising combiningthe at least part of the structure with a fluid and optically activeentities, wherein the optically active entities in the fluid can bind tothe at least part of the structure and wherein at least one of a bindingproperty of the optically active entities in respect of the at leastpart of the structure depends on the force acting on the at least partof the structure; and an optical property of optically active entitiesbound to the at least part of the structure depends on the force actingon the at least part of the structure.
 17. The method of claim 1,wherein the structure is a DNA molecule.
 18. The method of claim 1,wherein the optically active entities are DNA intercalator molecules.19. A controller, the controller being configured to perform the stepsof: controlling a light-sensitive system, e.g. a fluorescencemicroscope, to determine light information based on light from thestructure, the light being incident on at least a part of the lightsensitive system, wherein the at least part of the structure comprisesoptically active entities, such as DNA intercalator molecules, whereinat least one of an optical activity of the entities and a quantity ofthe entities depends on the force acting on the at least part of thestructure and wherein the light information defines a light propertyvalue associated with said at least part of the structure; anddetermining the force acting on the at least part of the structure onthe basis of said light property value and a reference light propertyvalue.
 20. The controller of claim 19, wherein the light-sensitivesystem is a fluorescence microscope.